Research ArticleIn Vitro Conservation of Some Threatened and EconomicallyImportant Ferns Belonging to the Indian Subcontinent

Shastri P. Shukla1 and P. B. Khare2

1 Plant Tissue Culture Laboratory, Plant Biotechnology Division, Central Institute of Medicinal and Aromatic Plants (CIMAP),Council of Scientific and Industrial Research, P.O. CIMAP, Lucknow 226015, India

2 Pteridology Laboratory, National Botanical Research Institute (NBRI), Council of Scientific and Industrial Research,Rana Pratap Marg, Lucknow 226001, India

Correspondence should be addressed to Shastri P. Shukla; sp shukla2002@yahoo.co.in

Received 1 May 2014; Revised 17 June 2014; Accepted 26 June 2014; Published 10 July 2014

Academic Editor: Curtis C. Daehler

Copyright © 2014 S. P. Shukla and P. B. Khare. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

This study was undertaken to identify methods of mass multiplication for five ornamental, economically important ferns(Nephrolepis biserrata (Sw.) Schott., N. cordifolia cv. “duffii” (L.) Presl., N. exaltata cv. bostoniensis (L.) Schott., Pteris vittata L.,and Cyclosorus dentatus Link.,) and three threatened ferns, namely, Cyathea spinulosa Wall. ex. Hook, Pityrogramma calomelanos(L.) Link., andMicrosorum punctatum (L.) Schott., through in vitro techniques. Collections were made from different biodiversityzones of India including Northeast Himalayas, Kumaon Himalayas, and Western Ghat and successfully introduced and grown ina fern-house. Aseptic cultures were raised at the morphogenic level of callus, axillary shoot, multiple shoot, and rooted plants. Anoptimized medium is described for each fern species. Plantlets were also produced from spore culture of Cyathea spinulosa andsuccessfully hardened under fern house conditions.

1. Introduction

Pteridophytes constitute an important part of the plantkingdom, as they are worldwide in distribution, with 12,000species distributed in a wide range of habitats. Pteridophyteshave enormous economic potentiality asmedicine, sources offood, fodder, fibre, flavouring agents, aromatic oil, perfume,dyes, and folk remedies [1–3]. In recent times ferns have alsoproved themselves valuable as pollution indicators, as well asinsect repellents, and some species have the ability to hyper-accumulate noxious metals and metalloids, making themgood phytoremediators [4–6]. In addition to their popularityas ornamental plants in the cut flower industry, pteridophyteshave been employed inAyurvedic,Homoeopathic, andUnanisystems ofmedicine since the times of Charaka and Sushruta.The aesthetic appeal and exquisite foliage patterns makethem popular plants for landscaping. According to a recentagricultural and industrial survey [7], the world floriculturetrade of cut flowers, green plants, and cut greens in 1995 was

US $ 6.8 billion, of which 55 percent were cut flowers, 37percent green plants, and 8 percent cut greens.

Due to overexploitation, habitat destruction, and otherbiotic interferences, numerous pteridophytic species arethreatened and some are critically endangered. Bir [8] iden-tified 100 species as threatened from throughout India, butlittle is known about the conservation status of many of theferns, and only 33 of those species have been categorized inred books. Chandra and Khare [9] enumerated 58 speciesas threatened, out of which 18 taxa are endangered from theKumaon Himalayas (India) region only.

Ferns are conventionally propagated both by sexual aswell as vegetativemethods.The sexualmethod of propagationinvolves raising plants from spores, whereas the vegeta-tive method involves propagation of specialized vegetativeorgans, such as bulbils, proliferous frond tip, aerial growths,stolon and tubers, offsets, gemmae, stipules, and root buds.These methods require more time and are comparativelyslow. In India, in comparison to angiosperms, few attempts

Hindawi Publishing CorporationJournal of BotanyVolume 2014, Article ID 949028, 8 pageshttp://dx.doi.org/10.1155/2014/949028

2 Journal of Botany

have been made on in vitro studies in ferns and fern-allies.Vascular cryptogams, especially ferns, have not been veryfavourable material for tissue culture because their vascularsystem is made up of highly differentiated tissues that aredifficult to proliferate into cell masses capable of growth invitro [10]. Although different workers have tried to initiatein vitro studies on ferns, very little work on tissue culture ofpteridophytes is reported [11].

The present investigation deals with collection and invitro and ex situ conservation of eight economically impor-tant and threatened taxa of pteridophytes belonging to differ-ent biodiversity zones of the Indian subcontinent.These fernsare economically important, andC. spinulosa, P. calomelanos,andM. punctatum are listed in Red Data Book as threatenedspecies, so the work presented here would be beneficial forthe biological conservation of threatened ferns as well ascommercial production of ornamental ferns through in vitrotechniques.

2. Materials and Methods

2.1. Collection of Plant Material and Type of Explants Used. Invitro studies were undertaken on the following fern species:Pteris vittata L.,Nephrolepis biserrata (Sw.) Schott.,N. cordifo-lia cv. duffii (L.) Presl.,N. exaltata cv. bostoniensis (L.) Schott.,Cyathea spinulosa Wall. ex Hook., Microsorum punctatum(L.) Copel., Cyclosorus dentatus Link., and Pityrogrammacalomelanos (L.) Link. Among these fern species, P. vittata,N. biserrata,N. cordifolia cv. duffii,N. exaltata cv. bostoniensis,M. punctatum, and C. dentatus were collected from differentphytogeographic region of India and successfully grownin the Fern House of CSIR-NBRI, Lucknow. Spores of C.spinulosa and P. calomelanos were collected from Didihat, inthe Pithoragarh district of KumaonHimalayas (Uttaranchal),Northeast region and Kodaikanal, Tirunelveli Tamilnadu,respectively (Figure 1; Table 1).

In the present study, explants from leaf primordia (0.5–1 cm.) from the fresh plant of P. vittata, N. cordifolia cv. duffii,N. exaltata cv. bostoniensis, M. punctatum, and C. dentatuswere used, whereas, in case ofC. spinulosa and P. calomelanos,leaf primordium (0.5 cm) of in vitro-raised sporophyte wasused as explants. In the case of N. biserrata, stolon explantsobtained from the live plant population growing in theFernery of CSIR-NBRI, Lucknow.

2.2. Raising Aseptic Cultures and Multiplication. Explantswere first washed thoroughly for 1-2 hours under tap water.The explants were then disinfected with 50–75% ethanolfor a few seconds (depending upon the nature of explant)and subsequently surface was sterilized with HgCl

2solution

(0.1% w/v). Finally, repeated rinsing of the explants was donewith sterile double distilled water. Treatment time variedfrom plant to plant and from explant to explant. Stolonparts were treated for 5–10min, spore, leaf primordium,circinate part of young fronds and leaf primordium, procuredfrom in vitro-raised sporophytes, were treated for 2–5, 2–10, 5–10, and 1-2min, respectively. The explants were thenthoroughly washed with sterile double distilled water (3-4

Gangetic plain Northeast

BayofBengal

Pakistan

Deccanplateau

Tibet

Nepal

Lakshadweep island(India)

Nephrolepis biserrata

N. cordifolia cv.duffii

N. exaltata cv. bostoniensis Microsorium punctatum

Pityrogramma calomelanos

Cyathea spinulosa

Cyclosorus dentatus

Dessert

Kumaon

WesternHimalayas

Myanm

ar

Western Ghats

N

Pteris vittata

Semiar

id

Figure 1: Different biodiversity zones of Indian subcontinent fromwhere eight pteridophytic species were collected.

times). Sterilized explants were inoculated into the P&T, MS,and KnD media with at full, half, and one-fourth strengthwith different combinations of growth hormones along withvarious percentages of agar and sugar. More than 50 mediacombinations were tested for raising aseptic cultures of allthe fern species. According to the morphogenetic responserequired, medium recipes were made by using basal mediafortified with different ranges of 2, 4-D, IBA, IAA, KN, andNAA. Three replicate cultures for each treatment were incu-bated in a culture room under 38𝜇 mol m−2 s−1 fluorescentlight for 15 hrs per day. Temperature and humidity of theculture room were maintained at 25 ± 2∘C and about 60–70percent RH, respectively. The period of incubation usuallyvaried from 30 to 45 days according to the need of theexperiment. Cultures were raised in 19.5 cm long × 3.5 cmdiameter and 15 cm long × 2.5 cm diameter culture tubes aswell as in Petri dishes of 100mm× 17mmand 80mm× 17mmsize.

2.3. Fern House Hardening. In vitro regenerated plantlets,growing in aseptic cultures, were rooted successfully in theliquid P&Tmedia [12].The rooted plantlets were acclimatized

Journal of Botany 3

Table 1: Details of the pteridophytic plants collected, importance, and their site of collection for in vitro conservation.

S. number Pteridophytictaxa Family Part used Conservation

status Importance Zone ofcollection

1 Nephrolepisbiserrata Nephrolepidaceae Whole plant —

Ornamental fern, muchdemand in nursery traitand cut flower industry.Cultivated in thebotanical gardens asornamentals and aspotted plants for indoordecoration.

Gangetic plain

2 N. cordifolia cv.L duffii Nephrolepidaceae Whole plant,

young leaves —Ornamental fern. Youngleaves are cooked asvegetable by the tribals.

Gangetic plain

3 N. exaltata cv.bostoniensis Nephrolepidaceae Whole plant,

young leaves —

Ornamental fern, muchdemand in nursery traitand cut flower industry.Cultivated in thebotanical gardens asornamentals and aspotted plants for indoordecoration.

Gangetic plain

4 Cyatheaspinulosa Cyatheaceae Trunk and

pith Threatened

A threatened tree fernlisted in Red-Data Book.Trunk is extensivelyusing in the orchidcultivation. Pith fromthe trunks is used as afood product

Kumaon,NortheastHimalaya

5 Pityrogrammacalomelanos Hemioitidaceae Whole plant Threatened

A threatened tree fernlisted in Red-Data Book.It is hyperaccumulator ofarsenic

Tamilnadu

6 Microsoiumpunctatum Polypodiaceae Fronds Threatened

A threatened tree fernlisted in Red-Data Bookand a popularhouseplant. Muchdemand in nursery traitas an economicallyimportant fern due to itsedible value in Asia andPacific region. Thecooked fronds of thisfern are consumed inNew Guinea.

WesternHimalayas

7 Pteris vittata Pteridaceae Whole plant —

An ornamental fern.Hyperaccumulator ofarsenic. In Florida, USAit is using as aphytoremediator inarsenic-enriched soil.

Gangetic plain

8 Cyclosorusdentatus Thelypteridaceae Whole plant — An ornamental fern Gangetic plain

in a solution consisting of only the inorganic salts of the sameP&T media, before their final transplantation to potted soil.The plantlets alongwith their roots were carefully taken out ofthe Petri dishes with the help of forceps. Roots of the plantlets

were thoroughly washed under running tap water to removethe entire adhering nutrient agar. After rinsing with distilledwater, they were acclimatized in inorganic salt solution atleast for a period of ca. 20 days. The nutrient solution was

4 Journal of Botany

Table 2: Morphogenic level along with the optimised medium and glasshouse hardening status of 8 plant species processed for in vitroconservation.

S. No Plant species Explant used Callus (GR inmgL−1)

Cultures established at thelevel of axillary/multipleshoots (GR in mgL−1)

Rooted plants(GR in mgL−1)

Numberof

replicates

Time taken/success rateduring

hardening

1 Nephrolepisbiserrata Stolon — P&T + 2,4-D (2.0) + NAA

(2.0) P&T + IAA (1.0) 10 2wks/100%

2 N. cordifolia cv.duffii Leaf primordium — P&T + 2,4-D (2.0) + BAP

(2.0)P&T + NAA (2.0)+ Kn (1.0) 10 3-4wks/60%

3 N. exaltata cv.bostoniensis Leaf primordium — MS + 2,4-D (1.0) + BAP

(0.5) — 10 —

4 Cyatheaspinulosa

Leaf primordiumprocured from invitro-raisedsporophytes

KnD + 2,4-D (2.0) P&T + BAP (2.0) + NAA(1.0) P&T + IBA (1.0) 10 3-4wks/100%

Spore — P&T basal media P&T basal media 10 3-4wks/80%

5 Pityrogrammacalomelanos

Circinate part ofyoung fronds KnD + 2,4-D (1.0) — — 10 —

Spore — P&T basal media P&T basal media 10 5–7wks/70%

6 Microsorumpunctatum

Leaf primordium1/4 strength of MSmedium(Hormone- free)

— — 10 —

Spore — P&T basal media P&T basal media 10 5–7wks/50%

7 Pteris vittata Leaf primordium

MS + 2,4-D(2.0) + BAP(0.5)GGBs-MS + 2,4-D(0.5) + BAP (0.5)

MS + IAA (0.5)+ BAP (2.0)

P&T + NAA (1.0)+ BAP (0.5) 10 3-4wks/100%

8 Cyclosorusdentatus Leaf primordium — P&T + BAP (1.0) + IAA

(0.5) 10

periodically changed every 5 days while the root system ofplantlets was also thoroughly washed with double distilledwater.

The acclimatized plants were transplanted in smallearthen pots containing a mixture of soil and leaf mouldin the ratio of 2 : 1. The earthen pots along with the pottingmixture were sterilized by autoclaving at 0.7 kg/cm2 pressuresfor 10min. before transplantation. The plants were initiallycovered with polyethylene chambers for a period of 4 to 8days after transfer in liquid as well as in potted soil to preventthem fromdesiccation. Plants of two species (C. spinulosa andN. biserrata) were later grown under Fern House conditionsto observe their performance (Table 2).

3. Results and Discussion

Eight threatened and economically important ferns weresuccessfully established in vitro at various morphogeniclevels. Details pertaining to optimized media combinationand morphogenic levels are given in Table 2. C. spinulosaand P. vittata showed both induction and proliferation ofcallus as well as shoots. P. calomelanos and M. punctatumwere established only at the callus level, while N. exaltata cv.

“bostoniensis” and C. dentatus were maintained only at shootlevel. An efficient in vitro protocol has been standardizedfor the commercial production of an ornamental fern, N.biserrata through stolon explants. A threatened tree fern,C. spinulosa, has been multiplied by two methods: throughcaulogenesis from leaf primordium explants procured fromin vitro-raised sporophyte and direct spore culture ([13];Figure 2). Leaf primordium explants obtained from freshplants growing in Fernery of NBRI, Lucknow, were used andevaluated for their regenerative potentiality in vitro in caseof P. vittata, M. punctatum, and C. dentatus. In case of P.vittata leaf primordium explants showed better response forthe callusing with GGBs andmultiple shoots, which had beentried for the first time.

All the shoot cultures showed first bud break within 20–30 days, except for C. spinulosa, which required a longertime (three months) to show first bud emergence. Out ofthese cultures, friable callus of only C. spinulosa and P. vittatagave rise to differentiation of multiple shoots. Cultures of N.cordifolia cv. “duffii” andN. exaltata cv. “bostoniensis” showedexcessive leaching of phenolics in themedium; therefore, theywere subcultured frequently. Supplementation of 40mg/Iascorbic acid in the respective media of N. cordifolia cv.“duffii” and N. exaltata cv. “bostoniensis” was able to control

Journal of Botany 5

(a)

(b) (c)

(d) (e)

(f) (g)

(h) (i)

(j)

(l) (m) (n) (o)

(k)

Figure 2: In vitro established cultures of some threatened and economically important ferns. (a) Regeneration ofNephrolepis biserrata throughstolon explants, (b) acclimatization of the in vitro-raisedplantlets of N. biserrata, (c) transplantation of the plantlets of N. biserrata in ferneryof CSIR-NBRI, Lucknow, (d) organogenesis in N. cordifolia cv. “duffii,” (e) mass multiplication of Cyathea spinulosa through spore culture,(f) mass multiplication of C. spinulosa through spore culture via caulogenesis through excised pieces of sporophytes, (g) transplantation ofin vitro-raised plantlets of C. spinulosa, in the fern house, (h) callus induction in Pteris vittata, (i) regeneration of leaflet in the callus cultureof P. vittata, (j & k) in vitro-raised plantlets of P. vittata growing in the fern house condition, (l) GGBs in P. vittata culture, (m) callus cultureof Pityrogramma calomelanos, (n) organogenesis in Cyclosorus dentatus, and (o) organogenesis inMicrosorum punctatum.

6 Journal of Botany

the leaching and browning to certain extent, but frequentsubculturing was the only feasible option observed in theseplant species. Characteristic GGBs formation was observedwithin 15 days in P. vittata cultures.

The shoots of N. biserrata, P. vittata, and C. spinulosawere highly proliferating. P. vittata and C. spinulosa showed100% germination rate of spore culture while only 50% and70% spore germination was observed in M. punctatum andP. calomelanos, respectively. Rooting was observed in N.biserrata, N. cordifolia cv. “duffii,” C. spinulosa, and P. vittata.Rooted plants were successfully transferred to the fern housewith a success rate of 60–80% (N. cordifolia cv. “duffii” and C.spinulosa) and 100% (N. biserrata and P. vittata) (Table 2).

In the present study, the morphogenetic potentialities ofvegetative explants of three Nephrolepis species, namely, N.biserrata, N. cordifolia cv. duffii, andN. exaltata cv. bostonien-sis were studied with a view to primarily develop methods oforganogenesis and, subsequently, production of plantlets. Inthe case ofN. biserrata, the explants of stolons were employedto induce differentiation of regenerants in them. In the courseof investigation, the proper combinations and balances ofgrowth hormones with appropriate inorganic salt mediawere enumerated for direct differentiation of regenerantsfrom explants. Regeneration of plants from explants withoutintervening callus formation was reported to mostly producegenetically identical plants, in contrast with the formationof plants via callus, in which case, the genetically variableplants may be produced [14–17]. The three Nephrolepisspecies differed in their regenerative potentialities, whichdepended on the nature of explants used.The stolon explantsused in the case of N. biserrata were highly regenerative,whereas leaf primordium explants of N. cordifolia cv. “duffii”and N. exaltata cv. “bostoniensis” were comparatively lessregenerative. This may be due to higher meristematic tissueactivity in stolon explants in comparison to leaf primordiumexplants. Survey of the literature revealed very few reportsof economically important ferns that have been multipliedthrough in vitro method. The most prominent example ofmass multiplication of an economically important fern is N.exaltata [18] in which callus induction was reported fromprovascular tissue of terminal and lateral buds of stolontips. Some other reports are available on other species ofNephrolepis, N. cordifolia [19–21], and N. exaltata [14, 22–25], on morphogenetic studies pertaining to the microprop-agation on a small scale. In vitro-regenerated plants had tobe acclimatized in an inorganic salt solution for about twoweeks in order to get 100 percent transplantation success insoil. The liquid culture phase for acclimatization of in vitro-raised plants was also necessary before transplantation in soilin case of N. biserrata [26], whereas in case of Cheilanthesviridis andDiplazium cognatum andMatteuccia struthiopterisdirect plantlet transplantation in soil was successful [27, 28],whichmay be due to tropical rain condition of the area, whichwas not prevailing in other areas including the present study,where a liquid culture phase was essential for acclimatization.In N. biserrata, when NAA was substituted with BAP with2,4-D (2mgL−1), shoot regeneration in stolon explants wasaugmented, and this was also more effective than othersynthetic auxin. Reports are available on the effects of Kn

and NAA on in vitro shoot multiplication and rooting in N.exaltata cv. “bostoniensis” through runner tip culture [29].These reports clearly indicated that Kn in combination withlow concentration of NAA (0.5–1mgL−1) produced shoots.The present investigation was carried out to identify the mostappropriate culture media for in vitro mass propagation ofthree species ofNephrolepis, namely,N. biserrata, N. cordifoliacv. “duffii,” andN. exaltata cv. “bostoniensis,” because no suchinvestigation has been reported to date. Some cultivars of thistaxon are sterile [30], and economically very significant; thus,propagation through tissue culture technique is required.

Callus was induced from explants of leaf primordial of P.vittata in order to utilize it formorphogenetic studies. In fernsgenerally the nutritional requirements for callus inductionand differentiation have been reported to be simple [31]. Cal-lus production and its differentiation from leaf primordiumis a comparatively difficult task because the percentage ofcontamination is high in the explants of young circinatefronds owing to their coiled pubescent nature. KshirsagarandMehta [32] reported induction and growth of callus fromrhizome segments ofP. vittata on “W”medium supplementedwith 2, 4-D alone. In the present study, 2, 4-D used aloneat various concentrations failed to induce callusing fromexplants of leaf primordia. However, 2, 4-D used with BAPwas most effective for induction and fast growth of callusat its higher concentration, whereas its low concentrationproduced a green, compact, and globular callus indicatingformation of GGBs. In vitro callusingwas achieved in in vitro-raised frond tip explants of P. calomelanos andM. punctatumon full-strength of KnD medium supplemented with 2,4-D(1mgL−1) and one-fourth strength of MS medium withouthormone, but differentiation of callus was not achieved,whereas, in the cases of N. exaltata cv. bostoniensis and C.dentatus, regeneration of shoots took place on full-strengthMS medium supplemented with 2,4-D (2mgL−1) and BAP(0.5mgL−1) and full-strength P&T medium supplementedwith BAP (1mgL−1) and IAA (0.5mgL−1), respectively. Dur-ing the course of investigation these taxa showed recalcitrantcharacteristics on in vitro studies (Figure 2).

4. Conclusion

In vitro studies on ferns and fern-allies are very meagrein comparison to angiospermic plants. Through the presentstudy, economically valuable and endangered ferns wereassessed for in vitro studies, which may help for mass-multiplication. Several pteridophytic plants are under thethreat of overexploitation and biodiversity depletion. Thereis urgent need of their ex situ conservation. Collection andin vitro cloning and conservation of the 8 economicallyimportant ferns in the present study open fresh avenuestowards the conservation and resource management of theoverexploited pteridophytic plants.

Abbreviations

BAP: 6-BenzylaminopurineCSIR: Council of Scientific and Industrial Research

Journal of Botany 7

2,4-D: 2,4-dichlorophenoxy acetic acidGGBs: Green globular bodiesGR: Growth regulatorHgCl2: Mercuric chloride

IAA: Indole-3-acetic acidIBA: Indole-3-butyric acidKn: KinetinKnD: Knudson mediumMS: Murashige and Skoog mediumNAA: 𝛼 Naphthalene acetic acidNBRI: National Botanical Research InstituteP&T: Parker &Thompson’s mediaW: White medium.

Conflict of Interests

The authors declare that they have no conflict of interests.

Authors’ Contribution

Shastri P. Shukla conceptualized and wrote the first draft ofthe paper; P. B. Khare helped in maintaining in vitro cultures.

Acknowledgments

The authors thank the Director of CSIR-NBRI, Lucknow,for providing the necessary facilities to carry out this work.Shastri P. Shukla is particularly thankful to the Departmentof Biotechnology, Government of India, New Delhi, forproviding the fellowship in the form of Senior Project Fellowduring the course of study (2003–2006).

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8 Journal of Botany

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